The proposed supplement project is a collaborative effort between Harvard and PNNL in response to the administrative supplement notice# NOT-ES-19-013. Due to their high surface area and diverse chemical functionality, 2D nanomaterials (2DNMs) have many potential important societal applications, including in the food industry, drug delivery, imaging and biosensing, and in agriculture practices. However, the potential health hazards of these emerging materials that may arise due to human exposure is poorly understood, least of all in the GI tract. Damage by 2DNMs to intestinal epithelium could cause local and systemic pathology, as well as disturbances in digestion and absorption of fluids, nutrients and toxins. Oral exposure to 2DNMs could alter the metabolic activities and community compositions of the gut microbiome, which in turn may impact human health. Our lack of understanding of the effects of emerging 2D ENMs on GI tract endpoints is an important knowledge gap. The objective of this collaborative effort is to develop, validate and employ an in vitro methodology that enables the systematic investigation of the effects of ingested 2DNMs on the gut epithelium and microbiome. Building on preliminary studies within the NHIR, Harvard and PNNL will collaborate to use an in vitro GI tract digestion system, combining a three-phase oral-gastric-small intestinal simulated digestion system, and a colonic microbiome reactor to address this knowledge gap through 3 aims: 1) characterize the interactions of ingested 2DNMs with food matrix, their physicochemical transformations across the GIT and potential effects on the small intestinal epithelium; 2) characterize the effects of ingested 2DNMs on the gut microbial populations; and 3) characterize the effects of ingested 2DNMs on the gut metabolome. The proposed supplement studies will provide the resources necessary to extend the NHIR consortium to include microbiome components associated with oral exposure routes for nanomaterials. We expect the success of this interdisciplinary work will provide the foundation for a novel new in vitro system useful for rapid nanomaterial hazard analyses, and which could be extended in future work to other types of particulates and environmental chemical exposures of importance to NIEHS missions.